EPO has shown robust neuroprotective effects in both in vitro and in vivo models of ischemic injury. Since less than 1% of systemically administered EPO crosses the blood brain barrier (BBB), large and multiple doses of EPO have been required to achieve effective concentrations in the brain. Such an administration regimen of EPO may lead to increases in hematocrit and stimulate the production of platelets, increasing the likelihood of microinfarctions and macroinfarctions, thus severely limiting or even precluding the use of EPO for stroke. Accordingly, alternate strategies to reduce erythropoietic activity of EPO and its potential side effects will greatly improve its clinical applications for the treatment of stroke. We will utilize two different approaches to overcome the potential side effects and delivery limitations of EPO. The first approach is the utilization of a mutant EPO (mEPO) lacking erythropoietic activity. We have recently successfully generated a novel EPO mutant that completlely lacks erythropoietic activity. Importantly, this mutant EPO remains its neuroprotective effect in both in vitro and in vivo models of ischemia with similar efficacies as wild-type EPO. The second approach is the use of the protein transduction technique to enhance the penetration of EPO crossing the BBB. Therefore, the objectives of this proposal are 1) to further test the neuroprotective effect of mutant EPO lacking erythropoietic activity in the clinically relevant middle cerebral artery occlusion (MCAO) model, and 2) to determine whether a more rapid delivery of protein transduction domain fused EPO or mEPO (EPO-TAT or mEPO-TAT) into brain can be translated into a decreased effective dose and a wider time-window of efficacy as compared to wild-type EPO. The following two Specific Aims are proposed: 1) To test the hypothesis that systemic administration of the mutant EPO (mEPO) lacking erythropoietic activity is neuroprotective against focal ischemic injury. These studies will determine: 1) whether mEPO reduces infarct volume in dose- and time-dependent manners without stimulating platelet and hematocrit;2) whether mEPO results in improved neurological outcomes;and 3) whether mEPO treatment activates the same cell survival pathways (PI3K/Akt, ERK1/2) in ischemic brain as wild-type EPO does. 2) To test the hypothesis that systemic administration of EPO-TAT or mEPO-TAT results in a more efficient delivery of the protein into the brain and offers a wider time window of efficacy compared to EPO alone. These studies will determine: A) whether EPO-TAT or mEPO-TAT offers same neuroprotective effects against ischemic injury at substantially lower doses as compared to the wild-type EPO, B) whether EPO-TAT or mEPO-TAT may provide a wider time window of efficacy compared to wild-type EPO and C) the effects of EPO-TAT or mEPO-TAT on inflammation following MCAO. PUBLIC HEALTH RELEVANCE: Erythropoietin (EPO) is a natural hormone for the maturation of red blood cells and has recently emerged as a promising candidate for neuroprotection in ischemic stroke. Large and multiple doses of EPO have been required to achieve effective concentrations in the brain due to the existence of the blood brain barrier (BBB), which allows only less than 1% of systemically administered EPO to cross the BBB. Such an administration regimen may lead to increased red blood cell and platelet production, making the blood more "sticky" and easily coagulated. We will utilize two different approaches to overcome the potential side effects and delivery limitations of EPO. The first approach is the utilization of a mutant EPO that protects the brain from ischemic injury, but lacks the ability to stimulate the production of red blood cell. Thus, this mutant EPO will make EPO therapy "safer" for stoke treatment. The second approach is the use of the protein transduction technique to enhance the penetration of EPO crossing the BBB. This novel approach will significantly decrease the amount of EPO required for treatment and possibly extend the treatment window. Taken together, this project will provide novel and safe approaches for the application of EPO as a therapeutic agent in stroke and in other neurological diseases.